Novel Ambient Oxidation Trends in Fingerprint Aging Discovered by Kendrick Mass Defect Analysis

A Kendrick mass defect (KMD) plot is an efficient way to disperse complex high-resolution mass spectral data in a visually informative two-dimensional format which allows for the rapid assignment of compound classes that differ by heteroatom content and/or unsaturation. Fingerprint lipid oxidation has the potential to be used to estimate the time since deposition of a fingerprint, but the mass spectra become extremely complex as the lipids degrade. We apply KMD plot analysis for the first time to sebaceous fingerprints aged for 0–7 days to characterize lipid degradation processes analyzed by MALDI-MS. In addition to the ambient ozonolysis of fingerprint lipids previously reported, we observed unique spectral features associated with epoxides and medium chain fatty acid degradation products that are correlated with fingerprint age. We propose an ambient epoxidation mechanism via a peroxyl radical intermediate and the prevalence of omega-10 fatty acyl chains in fingerprint lipids to explain the features observed by the KMD plot analysis. Our hypotheses are supported by an aging experiment performed in a sparse ozone condition and on-surface Paternò–Büchi reaction. A comprehensive understanding of fingerprint degradation processes, afforded by the KMD plots, provides crucial insights for considering which ions to monitor and which to avoid, when creating a robust model for time since deposition of fingerprints.

Pg 4  Table S2. Summary of heteroatom classes targeted in the study.
Pg 5 Table S3. Paternò-Büchi carbon-carbon double bond assignments of some TGs in fingerprints Pg 6 Supporting Figures (Pg 7-21) Figure S1. Original mass spectrum for fresh and 7-day aged fingerprint Pg 7 Figure S2. Overlain Kendrick plot for fresh and 7-day aged prints for the entire m/z and Kendrick mass defect range.
Pg 8 Figure S3. Monitoring the ambient laboratory variables over time.
Pg 9 Figure S5. Compound and heteroatom class annotated Kendrick plots.
Pg 14 Figure S9. KMD plot of 7-day aged print using a KMD base unit of 12 C1 16 O1.
Pg 15 Figure S10. Mass spectrometry images of select ions related to fingerprint oxidation.
Pg 17 Figure S13. Monitoring fingerprint lipid epoxide features during UVA aging experiment.
Pg 18 Figure S14. Monitoring fingerprint lipid epoxide features with different sample treatments.
Pg 18 Figure S15. Signal intensity profile of saturated DGs and the overlapped of TG, DG(A), and DG(E) series.
Pg 20 Figure S17. Example MS and MS/MS spectra of derivatized fingerprint triacylglycerols Pg 20 Figure S18. Compounds demonstrating the potential for time since deposition modeling.
Pg 21 Supporting References (Pg 21) S-3 approximately 5 μm, and ten laser shots in positive mode for the mass range of 100-1200 with a mass resolution setting of 30,000 at m/z 400 in the MS level. MS/MS was performed using the linear ion trap with a collision energy of 75 (arbitrary units) and an isolation width of 0.5-1.0 Da. The data was processed with Xcalibur.
Paternò-Büchi Data Analysis. Using benzophenone (BP) as a PB reagent for unsaturated lipids leads to a mass shift of 182.073 in the MS level ( Figure S17a and S17b), indicating oxetane ring formation through [2+2] photocycloaddition. The mass shifted m/z ratios can then be selected for MS/MS experiments to determine the double bond site as summarized in Table S3. MS/MS of BP-derivatized TG 48:2 is shown in Figure S17c as an example. It should be noted that fingerprint TGs are composed of multiple combinations of fatty acyl chains and we cannot differentiate sn-1 vs sn-2 position. In addition to the neutral loss of FA 16:0 (m/z 751.6) and FA 16:1 (m/z 753.6), the neutral loss of a BP-derivatized FA 16:1 is found at m/z 571.5, indicating TG 48:2 is mostly composed of TG 16:1/16:1/16:0. When oxetane ring fragments via retro-cycloaddition during MS/MS, it results in the loss of benzophenone at m/z 825.7 and the two characteristic fragments with Δm/z of 150.1, corresponding to the two orientations of benzophenone at the double bond site, "head-to-head" and "head-to-tail". The double bond site can be deduced from the neutral loss to the head-to-head peak being an aldehyde, CnH2nO. For example, the most abundant characteristic peaks for derivatized TG 48:2 are found at m/z 701.6 and 851.7, and the neutral losses correspond to n=10, thus ꞷ-10. The other set of diagnostic fragments are marked as solid fill yellow and purple boxes in Figure S17c indicating the presence of ꞷ-9 and ꞷ-8, respectively.

SUPPORTING DISCUSSION
Describing undefined plot features. There are series in the KMD plots that were not annotated with the list described and not obviously explained by ozonolysis. Further finding unassigned trends can be aided by dynamic normalization, which is similar to fractional base units described by Fouquet et al. 3 Figure  S7 is an example of this normalization where KM is equivalent to the product of the m/z and x, user specified number, divided by the IUPAC exact mass of the Kendrick base unit. This method changes the vertical dispersion of different homologous series, while maintaining the horizontal alignment within a homologous series. This can allow for the interpretation of closely aligned series observed when using static normalization alone. Using the dynamic KMD plot with x = 9, 13 C isotope series could be successfully separated and annotated in Figure S7. After accounting for 13 C isotopes, there are still some undefined series that need further investigation, some of which were investigated by the plots generated with different KMD base units. An example is using atomic oxygen, 16 O1 as a KMD base unit ( Figure S8). Though the information is also in the CH2 KMD plots, differences in oxygen content can easily be observed in this plot. We highlight two heteroatom classes that are more clearly observed in these plots. Including a series with high oxygen content in the fresh prints, which could be exogenous compounds (Figure S8c), and a heteroatom class of CcH2c-ZONa in the low mass range (m/z 250-300) which are tentatively assigned as unsaturated aldehydes ( Figure S8d). Aldehyde species are expected to result from ozonolysis but the resulting peaks are not in the expected mass range. Instead, they are comprised of 16 to 18 carbons and are present in the both the aged and fresh fingerprints. A KMD plot using 12 C1 16 O1 as a base unit helps detect a cluster of features present primarily in the 7-day aged plot, but these features are not likely to be associated with aging as they are near the S/N>30 cutoff and are also present in the fresh spectrum at a similar S/N ( Figure S9). Table S1. Summary of the environmental conditions during the aging.

SUPPORTING TABLES
Foot note: Values are calculated from Figure S3. Table S2. Summary table of searched heteroatom classes expected from ozonolysis. Table S2: 1. Not all searched heteroatom classes are found in KMD plot. "x" indicates searched theoretical heteroatom classes that were not found in the 7-day aged fingerprint in the lipid range for the KMD. 2. Several lipid substrates or their oxidation products belong to the same heteroatom class. The compounds within a heteroatom class are listed in order of expected relative signal contribution. 3. Z is an odd value for disodiated heteroatom classes and even otherwise. It increases by 2 for each DBE increase.  Foot notes for Table S3: a. Fragment ion pairs with an aldehyde fragment signal intensity greater than 10% of the most abundant aldehyde fragment (often the base peak) are considered in order to avoid over interpretation. The colors in TG 48:2 correlate with the fragments in the example MS/MS spectrum shown in Figure S17c.    . Kendrick mass defect bubble plots of the fresh (red) and 7-day old (black) fingerprints where bubble size is proportional to the relative abundance as seen in Figure S1. The legend is based on searched heteroatom class summarized in Table S2. Tentative compound assignments are provided for undegraded precursors, a single ozonolysis process, and a proposed epoxidation process.

Foot notes for
1. To minimize the clutter, only the first ozonolysis or epoxidation product is annotated; e.g., TG(AA) is not shown. The two Criegee ions in Scheme 1 (B and C product) cannot be distinguished when monosodiated and are labeled as '(B)', to simplify, instead of '(B/C)' as in Figure 2. Di-sodiated Criegee ion is only possible for carboxylic acid and labeled as '(C)'. 2. It is challenging to distinguish compounds within the same heteroatom class or those with very close KMDs. As long as they do not entirely overlap in the m/z dimension (e.g., Figure 3), such conflicts can be distinguished in a KMD plot. 3. Tentative compound assignments are made conservatively to minimize misinterpretation and a signal cutoff of S/N > 30 is used to avoid the overinterpretation of low intensity peaks. 4. Di-sodiated adduct of TG(C) (purple ◊) has very close KMD with the mono-sodiated TG(B) (black ◊). However, these series only overlap when interpreting three or more unsaturation in TG(B). Given that one of the double bonds in the original lipid must be consumed in the ozonolysis reaction to produce TG(B), the interference to the di-sodiated adduct profile is negligible. The same arguments can be made for the same ozonolysis products of the other lipid precursors. 5. Epoxides and aldehyde ozonolysis products have the same heteroatom class and cannot be distinguished. 6. We include 13 C1 and 13 C2 peaks in the KMD plots and include potassium adduct annotations to ensure that heteroatom class assignment is as accurate and transparent as possible. Some overlap is observed due to very close KMD with other compounds, but they are mostly distinguishable. Figure S6. Type-II isotopic overlap within the 7-day aged fingerprint spectrum demonstrating some limitations of the instrumentation at a higher m/z range, especially with drastic signal differences. Figure S7. Dynamic normalization KMD plot of the 7-day aged fingerprint with x=9.

S-13
Foot notes for Figure S7: 1. 13 C isotope contributions, up to three 13 C atoms for all searched m/z values, were included and annotated in this plot.
2. The dynamic normalization uses a different calculation for the KMD: Instead of x=14, the user inputs other values in order to manipulate the vertical dispersion of different homologous series while retaining the horizontal alignment within a homologous series. This allows visualizing homologous series that are nearly overlapped with static, x=14, normalization.
3. Note that a majority of the plot features are defined. Most of the unassigned features are well separated in the x=14 plot, and are aliased into the KMD range of the defined lipids when dynamic normalization is used (features circled red). However, this plot serves to check to ensure homologous series are not overlooked. The relationships of heteroatom compositions of unassigned features can be aided with using a different KMD base, as described in Figure S11 and S12.
4. The features circled in blue are tentatively assigned as sodiated adducts of unsaturated aldehydes, CcH2c-ZONa (Zmin = 2), and those circled in green as sodiated adducts of monoacylglycerols, CcH2c-ZO4Na (Zmin = 0), where assignments were assisted by using a KMD base of O in Figure S11.
5. The features circles in purple are an unassigned heteroatom classes that differ by CO, as seen in Figure  S12.
6. The features circles in orange are an unassigned heteroatom class with exact mass consistent with CcH2c-ZO4 (Zmin = 2), as protonated adducts.
7. Note the misassignment of the CcH2c-ZO7Na features as the 13 C isotope peaks. This is an obvious misassignment considering the lack of monoisotopic peaks.